Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that commonly causes ocular and pulmonary infections, as well as burn wound infections. This bacterium possesses an inherent resistance to most antibiotics, and is able to form biofilms that further enhance antibiotic resistance and chronic infections (Davies & Hilton (2009) Respir. Care 54:628-40). Of particular clinical importance is the prominence of P. aeruginosa infection in patients with compromised pulmonary function. By the age of 18, 80% of all patients with cystic fibrosis (CF) have a chronic P. aeruginosa lung infection (Geller (2009) Respir. Care 54:658-70). Furthermore, chronic obstructive pulmonary disorder (COPD) is the fourth leading cause of death world-wide (Vogt, et al. (2009) S D Med. Spec No 30-7), and P. aeruginosa pulmonary infection in these patients results in a rapid decline in lung function and a poor long-term prognosis (Murphy, et al. (2008) Am. J. Respir. Crit. Care Med. 177:853-60). P. aeruginosa also causes many nosocomial infections, exacerbating ventilator-associated pneumonias and hospital-acquired pneumonia (Zavascki, et al. (2006) Crit. Care 10:R114). Recently, a more aggressive strain of P. aeruginosa emerged in Liverpool, England that caused an epidemic in the CF community (Salunkhe, et al. (2005) J. Bacteriol. 187:4908-20). Therefore, finding new and effective ways to prevent and treat P. aeruginosa infection will help to reduce human morbidity and mortality.
During the course of infection, P. aeruginosa produces and secretes an arsenal of toxins and virulence factors (Kipnis, et al. (2006) Med. Mal. Infect. 36:78-91; Bleves, et al. (2010) Int. J. Med. Microbiol. 300:534-43). Of particular interest is the virulence factor Cif (Cystic fibrosis transmembrane conductance regulator Inhibitory Factor), an epoxide hydrolase (EH) that enters human cells and prevents the deubiquitination of the cystic fibrosis transmembrane conductance regulator (CFTR)(Bomberger, et al. (2011) PLoS Pathog 7:e1001325). Patients with CF have a mutation in the chloride ion channel CFTR that prevents its function and/or localization to the apical surface of airway epithelial cells, resulting in an osmotic imbalance that dehydrates the airway surface liquid and prevents mucociliary clearance (Rogan, et al. (2011) Chest 139:1480-90). Cif induces a rapid decline in cell surface CFTR levels (MacEachran, et al. (2007) Infect. Immun. 75:3902-12), essentially phenocopying the genetic disorder CF (Swiatecka-Urban, et al. (2006) Am. J. Physiol. Cell Physiol. 290:C862-72). In patients with wild-type CFTR, Cif maintains a persistent infection. In patients with CF that have a P. aeruginosa infection, Cif could greatly impede the efficacy of therapies designed to rescue CFTR function. Cif has also been shown to affect other ABC transporters (Ye, et al. (2008) Am. J. Physiol. Cell Physiol. 295:C807-818), suggesting that it may have additional deleterious effects on cellular physiology in vivo.
Cif is the first reported example of an epoxide hydrolase utilized as a bacterial virulence factor (Bahl, et al. (2010) J. Bacteriol. 192:1785-95). Cif possesses the hallmark catalytic triad that is characteristic of α/β hydrolases, which includes a nucleophile and a charge relay His and acid (Holmquist (2000) Curr. Protein Pept. Sci. 1:209-235). Prior to structural elucidation, Cif's catalytic triad His was predicted to be at position 269 by sequence alignment, and this residue was mutated to Ala (MacEachran, et al. (2007) supra). Cif-H269A was found lacking in enzyme activity using the colorigenic EH substrate S-NEPC. This mutant protein was also shown to be deficient in lowering apical surface CFTR abundance of human cells, suggesting a link between EH enzyme activity and the cellular effects of Cif. However, when the structure of Cif was determined by X-ray crystallography, it became clear that His297 was in fact the catalytic triad His (Bahl, et al. (2010) J. Bacteriol. 192:1785-1795). The catalytic triad of Cif is buried within the core of the protein at the interface between the cap and core domains. However, His269 is located on the protein surface, and appears to be positioned at the mouth of the tunnel leading to the active site.
To analyze the Cif effect, an understanding of how Cif functions as an EH is needed. EHs are an extensively studied class of enzymes (Arand, et al. (2005) Methods Enzymol. 400:569-588; Arand, et al. (2003) Drug Metab. Rev. 35:365-383; Morisseau & Hammock (2005) Annu. Rev. Pharmacol. Toxicol. 45:311-333). The active site is sequestered within the interior of the protein, at the interface between the α/β hydrolase core domain and a cap domain. According to the canonical mechanism, an epoxide substrate enters the active site and is bound by a ring-opening pair of polar residues. A nucleophile attacks an epoxide carbon, opening the ring and forming a covalent intermediate (Pinot, et al. (1995) J. Biol. Chem. 270:968-7974). Further, a charge-relay His-acid pair activates a water molecule to nucleophilically attack the enzyme-substrate intermediate and release the hydrolysis product. While Cif exhibits multiple sequence and structural deviations from the archetypal EH active site (Bahl & Madden (2012) Protein Pept. Lett. 19:186-193; Bahl, et al. (2010) supra), their comparison nonetheless allows for a focused analysis.